Effect of growth media and phase on Raman spectra and discrimination of mycobacteria

When developing a Raman spectral library to identify bacteria, differences between laboratory and real world conditions must be considered. For example, culturing bacteria in laboratory settings is performed under conditions for ideal bacteria growth. In contrast, culture conditions in the human body may differ and may not support optimized bacterial growth. To address these differences, researchers have studied the effect of conditions such as growth media and phase on Raman spectra. However, the majority of these studies focused on Gram‐positive or Gram‐negative bacteria. This article focuses on the influence of growth media and phase on Raman spectra and discrimination of mycobacteria, an acid‐fast genus. Results showed that spectral differences from growth phase and media can be distinguished by spectral observation and multivariate analysis. Results were comparable to those found for other types of bacteria, such as Gram‐positive and Gram‐negative. In addition, the influence of growth phase and media had a significant impact on machine learning models and their resulting classification accuracy. This study highlights the need for machine learning models and their associated spectral libraries to account for various growth parameters and stages to further the transition of Raman spectral analysis of bacteria from laboratory to clinical settings.      

Evaluation of sample preparation methods for MALDI-TOF MS identification of highly dangerous bacteria

Aims: To propose a universal workflow of sample preparation method for the identification of highly pathogenic bacteria by MALDI‐TOF MS. Methods and Results: Fifteen bacterial species, including highly virulent Gram‐positive (Bacillus anthracis and Clostridium botulinum) and Gram‐negative bacteria (Brucella melitensis, Burkholderia mallei, Francisella tularensis, Shigella dysenteriae, Vibrio cholerae, Yersinia pestis and Legionella pneumophila), were employed in the comparative study of four sample preparation methods compatible with MALDI‐TOF MS. The yield of bacterial proteins was determined by spectrophotometry, and the quality of the mass spectra, recorded in linear mode in the range of 2000–20 000 Da, was evaluated with respect to the information content (number of signals) and quality (S/N ratio). Conclusions: Based on the values of protein concentration and spectral quality, the method using combination of ethanol treatment followed by extraction with formic acid and acetonitrile was the most efficient sample preparation method for the identification of highly pathogenic bacteria using MALDI‐TOF MS. Significance and Impact of the Study: The method using ethanol/formic acid generally shows the highest extraction efficacy and the spectral quality with no detrimental effect caused by storage. Thus, this can be considered as a universal sample preparation method for the identification of highly virulent micro‐organisms by MALDI‐TOF mass spectrometry.     

Regulatory mutants of the Vibrio harveyi lux system

Regulatory mutants of the luminescent bacterium, Vibrio harveyi, have been isolated whose light emission can be stimulated by extracts of the growth media. Chloroform extracts of conditioned media in which V. harveyi has been grown can increase light emission in one of the dark mutants, D34, over 10³-fold. An increase in the level of the mRNA and the enzymes associated with the lux system can also be demonstrated. Analysis of the expression of the lux system in Escherichia coli transformed with DNA from the D34 regulatory mutant demonstrates that the mutation resides outside the luciferase structural genes. The results suggest that the decrease in light emission in the regulatory mutants may be due to a mutation in synthesis of an autoinducer analogous to that found for the Vibrio fischeri lux system.     

Highly fluorescent carbon dots from wheat bran as a novel drug delivery system for bacterial inhibition

In this study, we prepared carbon dots (CDs) from wheat bran via hydrothermal treatment at 180°C for 3 h. The prepared CDs showed blue‐green fluorescence under UV light. The fluorescence emission study of the CDs revealed that they showed maximum fluorescence emission at 500 nm. The prepared CDs showed a high quantum yield of 33.23%. Solvent‐dependent fluorescence emission analysis of the CDs was performed to study the variation in fluorescence emission characteristics with solvent polarity. The prepared CDs were conjugated with amoxicillin (AMX) to explore its potential for use as a drug delivery agent for AMX. The drug release profile of the CD–AMX conjugates was analyzed at different pH (5.0, 6.8 and 7.2) to study drug release kinetics. CD–AMX conjugates showed notable bacterial inhibition against Gram‐positive (S. aureus) and Gram‐negative (E. coli) strains with minimal cytotoxic effects, indicating its potential as a promising antibacterial drug delivery system.     

Far‐red dependent changes in the chemical composition of Spirulina platensis

The influence of far‐red light (FRL) was studied on the chemical composition of Spirulina platensis biomass. The following light compositions were used during the culture white light, blue‐red LED light (BRL) and BRL supplemented with FRL (BRFRL). Chlorophyll and phenol contents were measured by spectrophotometric methods, whereas presence of carotenoids, lipids, and phycobiliproteins were estimated by Fourier‐transform Raman spectrometry. Additionally, phenol content was investigated by fluorescence intensity of algae culture in the range of 430–650 nm. The content of chlorophyll and phenols in algae cells depended on the spectral composition of light and was the highest under BRL (16.7 ± 0.5 and 9.1 ± 0.6, respectively). It was shown that there is a positive linear correlation (R = 0.902 at p < 0.0000001) between the ratio of relative fluorescence intensity of S. platensis suspensions at 450 nm to the suspensions at 540 nm (F450/F540) and the content of phenolic compounds in the biomass. Changes in the F450/F540 ratio can explain approximately 80% changes of phenol contents in algae cells. Spirulina platensis Raman spectra demonstrated that the biomass of algae growing under white light and BRL had a significantly higher intensity of phycobiliprotein bands than the algae growing under BRFRL.     

High‐yield recombinant expression of the chicken antimicrobial peptide fowlicidin‐2 in Escherichia coli

The antimicrobial peptide fowlicidin‐2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin‐2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin‐2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin‐2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET‐32a(+), which features fusion protein thioredoxin at the N‐terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria‐Bertani (LB) medium. After isopropyl‐β‐D‐thiogalactopyranoside (IPTG) induction, the fowlicidin‐2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse‐phase high‐performance liquid chromatography (RP‐HPLC), ～6.0 mg of fowlicidin‐2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram‐positive and Gram‐negative bacteria, and even drug‐resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large‐scale production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:369–374, 2015     

“Quorum sensing” regulation and the structure of <Emphasis Type="Italic">lux</Emphasis> the operon in marine bacteria <Emphasis Type="Italic">Aliivibrio logei</Emphasis>

A group of luminescent strains of marine bacteria Aliivibrio logei has been isolated (basins of the Okhotsk, White and Bering Seas). Strains A. logei were shown to be psycrophilic bacteria with an optimal growth temperature of approximately 15°C. Bioluminescent characteristics of strains were studied, and the expression of lux genes was shown to be regulated by the “quorum sensing” system. The A. logei lux operon was cloned in Escherichia coli cells and the structure of this operon and its nucleotide sequence were determined. The structure of A. logei lux operon differs markedly from that in the closely related species of luminescent marine bacteria A. fischeri. In the structure of the A. logei lux operon, the luxI gene is absent in front of luxC, and a fragment containing luxR2-luxI genes is located immediately after luxG gene. Luminescent psycrophilic marine bacteria of A. logei are assumed to be widely distributed in cold waters of northern seas.     

Synthesis of short cationic antimicrobial peptidomimetics containing arginine analogues

Worldwide efforts are underway to develop new antimicrobial agents against bacterial resistance. To identify new compounds with a good antimicrobial profile, we designed and synthesized two series of small cationic antimicrobial peptidomimetics (1–8) containing unusual arginine mimetics (to introduce cationic charges) and several aromatic amino acids (bulky moieties to improve lipophilicity). Both series were screened for in vitro antibacterial activity against a representative panel of Gram‐positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram‐negative (Escherichia coli and Klebsiella pneumoniae) bacterial strains, and Candida albicans. The biological screening showed that peptidomimetics containing tryptophan residues are endowed with the best antimicrobial activity against S. aureus and S. epidermidis in respect to the other synthesized derivatives (MIC values range 7.5–50 µg/ml). Moreover, small antimicrobial peptidomimetics derivatives 2 and 5 showed an appreciable activity against the tested Gram‐negative bacteria and C. albicans. The most active compounds (1–2 and 5–6) have been tested against Gram‐positive established biofilm, too. Results showed that the biofilm inhibitory concentration values of these compounds were never up to 200 µg/ml. The replacement of tryptophan with phenylalanine or tyrosine resulted in considerable loss of the antibacterial action (compounds 3–4 and 7–8) against both Gram‐positive and Gram‐negative bacterial strains. Furthermore, by evaluating hemolytic activity, the synthesized compounds did not reveal cytotoxic activities, except for compound 5. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Vibrio azureus emits blue-shifted light via an accessory blue fluorescent protein

Luminous marine bacteria usually emit bluish-green light with a peak emission wavelength (λ(max) ) at about 490?nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein (lumazine protein: LumP) that causes a blue shift, from λ(max) ≈?490 to λ(max) ≈?476?nm. However, the incidence of blue-shifted light emission or the presence of accessory fluorescent proteins in bacteria of the genus Vibrio has never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio azureus emit a blue-shifted light with a peak at approximately 472?nm, whereas other Vibrio strains emit light with a peak at around 482?nm. Therefore, we investigated the mechanism underlying this blue shift in V.?azureus NBRC 104587(T) . Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light emission spectrum of the strain. This result strongly suggested that VA-BFP was responsible for the blue-shifted light emission of V.?azureus.     

Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish,Kryptophanaron alfredi (family Anomalopidae) to other luminous bacteria based on bacterial luciferase (luxA) genes

Flashlight fishes (family Anomalopidae) have light organs that contain luminous bacterial symbionts. Although the symbionts have not yet been successfully cultured, the luciferase genes have been cloned directly from the light organ of the Caribbean species, Kryptophanaron alfredi. The goal of this project was to evaluate the relationship of the symbiont to free-living luminous bacteria by comparison of genes coding for bacterial luciferase (lux genes). Hybridization of a luxAB probe from the Kryptophanaron alfredi symbiont to DNAs from 9 strains (8 species) of luminous bacteria showed that none of the strains tested had lux genes highly similar to the symbiont. The most similar were a group consisting of Vibrio harveyi, Vibrio splendidus and Vibrio orientalis. The nucleotide sequence of the luciferase subunit gene luxA of the Kryptophanaron alfredi symbiont was determined in order to do a more detailed comparison with published luxA sequences from Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi. The hybridization results, sequence comparisons and the mol% G+C of the Kryptophanaron alfredi symbiont luxA gene suggest that the symbiont may be considered as a new species of luminous Vibrio related to Vibrio harveyi.The nucleotide sequence reported in this article has been deposited in Genbank under accession number M36597     

Visualization of bioluminescence as a marker of gene expression in rhizobium-infected soybean root nodules

The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.     

Effective inactivation of food pathogens Listeria monocytogenes and Salmonella enterica by combined treatment of hypericin-based photosensitization and high power pulsed light

Aims: The aim of this study was to evaluate the inactivation efficiency of Listeria monocytogenes ATC_L3C 7644 and Salmonella enterica serovar Typhimurium strain DS88 by combined treatment of hypericin (Hyp)‐based photosensitization and high power pulsed light (HPPL). Methods and Results: Cells were incubated with Hyp (1 × 10^?5 or 1 × 10^?7 mol l^?1) in PBS and illuminated with a light λ = 585 nm. For the combined treatment, bacteria were, after photosensitization, exposed to 350 pulses of HPPL (UV light dose = 0·023 J cm^?2). Fluorescence measurements were performed to evaluate optimal time for cell–Hyp interaction. Results indicate that Hyp tends to bind both Listeria and Salmonella. After photosensitization treatment, Listeria population was reduced 7 log, whereas Salmonella was inactivated just 1 log. Electron photomicrograps of Salmonella and Listeria confirmed that photosensitization induced total collapse of the Listeria cell wall, but not that of Salmonella. After combined photosensitization–HPPL treatment, the population of Listeria was diminished by 7 log and Salmonella by 6·7 log. Conclusions: Listeria can be effectively inactivated by Hyp‐based photosensitization (7 log), whereas Salmonella is more resistant to photosensitization and can be inactivated just by 1 log in vitro. Combined treatment of photosensitization and pulsed light inactivates effectively (6·7–7 log) both the Gram‐positive and the more resistant to photosensitization Gram‐negative bacteria. Significance and Impact of the Study: A new approach to combat Gram‐positive and Gram‐negative bacteria is proposed, combining photosensitization with high power pulsed light.     

In vitro activity of novel in silico‐developed antimicrobial peptides against a panel of bacterial pathogens

Antimicrobial‐peptide‐based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β‐sheet structure in 10‐mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram‐negative and 0.94 to 20.65 μM (1.72‐46.5 µg/ml) for Gram‐positive bacteria. Interestingly, in high‐salt environment, the antibacterial activity was generally maintained for Gram‐negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram‐negative bacteria. A linear time‐dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg²⁺ and Ca²⁺, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti‐infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram‐negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg²⁺ and Ca²⁺, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Investigation of the roles of T6SS genes in motility, biofilm formation, and extracellular protease Asp production in Vibrio alginolyticus with modified Gateway-compatible plasmids

Aims: The aims of this study were to create and evaluate the Gateway‐compatible plasmids for investigating the function of genes in Vibrio alginolyticus and other Gram‐negative bacteria. Methods and Results: In this study, Gateway‐compatible plasmids were successfully constructed for rapid and comprehensive function analysis of genes. Taking advantage of these plasmids, the in‐frame deletion mutant strains and their complemented strains of five T6SS genes, including dotU1, VEPGS_0008, VEPGS_0011, hcp2 and ppkA2, were obtained. The results illustrated that all the mutant strains showed no significant effects on extracellular protease production, expression of Hcp1, and biofilm formation when compared to the wild‐type strain, but in‐frame deletion of VEPGS_0008 resulted in obvious biofilm reduction and the complemented strain restored to the level of the wild‐type strain. Besides, in‐frame deletion of dotU1, VEPGS_0008 and ppkA2 abolished the swarming ability. Conclusions: A set of Gateway‐compatible vectors for internal insertion, in‐frame deletion and complementation of the target genes is constructed to facilitate the general and rapid function analysis of genes involved in T6SS in Vibrio alginolyticus. Significance and Impact of the Study: The modified Gateway‐compatible plasmids greatly facilitate the high‐throughput and convenient function analysis of the unidentified genes.     

Efflux pump‐deficient mutants as a platform to search for microbes that produce antibiotics

Pseudomonas putida DOT‐T1E‐18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild‐type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT‐T1E‐18 on solid Luria–Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT‐T1E‐18 growth was an isolate named 250J that, through multi‐locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram‐positive bacteria, Gram‐negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram‐positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin.     

Antibacterial activity of naringin derivatives against pathogenic strains

Aims: To study the antimicrobial activity of naringin (NAR), a flavonoid extracted from citrus industry waste, and NAR derivatives [naringenin (NGE), prunin and alkyl prunin esters] against pathogenic bacteria such as L. monocytogenes, E. coli O157:H7 and S. aureus. The relationship between the structure of the chemical compounds and their antagonistic effect was also analysed. Methods and Results: The agar dilution technique and direct contact assaying were applied. NGE, prunin and NAR showed no antimicrobial activity at a concentration of 0·25 mmol l^?1. Similarly, fatty acids with a chain length between C2 and C18 showed no antimicrobial activity at the same concentration. However, prunin‐6″‐O‐acyl esters presented high antibacterial activity, mainly against Gram‐positive strains. This activity increased with increasing chain length (up to 10–12 carbon atoms). Alkyl prunin esters with 10–12 carbon atoms diminished viability of L. monocytogenes by about 3 log orders and S. aureus by 6 log orders after 2 h of contact at 37°C and at a concentration of 0·25 mmol l^?1. The compounds examined were not effective against any of the Gram‐negative strains assayed, even at the highest concentration. Conclusions: Addition of sugars to the aglycone did not enhance its antimicrobial activity. Attachment of a saturated aliphatic chain with 10–12 carbon atoms to the A ring of the flavonoid (or to sugars attached to this ring), seems to be the most promising modification. In conclusion, alkyl prunin esters with a chain length of C10–C12 have promising features as antimicrobial agents because of their high antilisterial and antistaphylococcal activity. Significance and Impact of the Study: This study shows that it is possible to obtain NAR derivatives with important antimicrobial activity, especially against Gram‐positive pathogenic bacteria. It also provides guidelines on the structural modifications in similar molecules to enhance the antimicrobial activity.     

Stimulation of DNA repair as an evolutionary drive for bacterial luminescence

It was demonstrated recently that luminescence of a free‐living marine bacterium, Vibrio harveyi, stimulates DNA repair, most probably by activation of the photoreactivation process. Here, we ask whether the stimulation of DNA repair could be an evolutionary drive that ensured maintenance and development of early bacterial luminescent systems. To test this hypothesis, we cultivated V. harveyi lux⁺ bacteria and luxA mutants in mixed cultures. Initial cultures were mixed to obtain a culture consisting of roughly 50% lux⁺ cells and 50% luxA mutants. Then bacteria were cultivated for several days and ratio of luminescent to dark bacteria was measured. Under these conditions, luxA mutants became highly predominant within a few days of cultivation. This indicates that, without a selective pressure, the luminescence is a disadvantage for bacteria, perhaps due to consumption of significant portion of cell energy. However, when the same experiments were repeated but cultures were irradiated with low UV doses, luminescent bacteria started to predominate shortly after the irradiation. Therefore, we conclude that stimulation of photoreactivation may be an evolutionary drive for bacterial bioluminescence. Copyright © 2003 John Wiley & Sons, Ltd.     

Analogues of the frog skin peptide alyteserin‐2a with enhanced antimicrobial activities against Gram‐negative bacteria

The emergence of strains of multidrug‐resistant Gram‐negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin‐2a (ILGKLLSTAAGLLSNL.NH₂) is a cationic, α‐helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin‐2a while maintaining amphipathicity by the substitution Gly¹¹→ Lys enhanced the potency against both Gram‐negative and Gram‐positive bacteria by between fourfold and 16‐fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC₅₀ = 24 µm ). Antimicrobial potency was increased further by the additional substitution Ser⁷→Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC₅₀ = 38 µm ). However, the peptide containing d ‐lysine at positions 7 and 11 showed high potency against a range of Gram‐negative bacteria, including multidrug‐resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µm ) but appreciably lower haemolytic activity (LC₅₀ = 185 µm ) and cytotoxicity against A549 human alveolar basal epithelial cells (LC₅₀ = 65 µm ). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.